This application relates to plant sterol biosynthetic enzymes, genes, and their uses.
Plant sterols belong to a large group of secondary compounds known as terpenes or isoprenoids. Sterol biosynthesis in plants generally involves a series of different enzymatic steps in the isoprenoid pathway that result in the formation of a variety of sterol end products (Benveniste Ann. Rev. Biochem. 37:275, 1986). Although such sterol compounds have been identified in higher plants, their function in plant growth and development is poorly understood.
One such plant sterol, brassinolide, that belongs to a class of sterols referred to as brassinosterioids (BR), was first discovered in the pollen of Brassica napus (Grove et. al., Nature 281: 216, 1979). Brassinosteroids are growth-promoting natural products having structural similarities to animal steroid hormones. The wide distribution of brassinosteroids in the plant kingdom, their effect on cell proliferation and elongation, and their interactions with other plant hormones (e.g., cytokinins), have indicated that these compounds are plant-growth regulators. Brassinosteroids are thought to promote hypocotyl elongation, leaf unrolling, and xylem differentiation. In addition, such compounds are also believed to be involved in de-etiolation of cotyledons, root elongation, radial growth, and anthocyanin formation.
The function of plant sterol growth regulators, such as BR, in relationship to other classes of plant growth regulators such as auxin, gibberellin, abscisic acid, and cytokinin, during plant development also needs to be evaluated. For example, the growth regulator, cytokinin, is known to affect a variety of developmental processes including photomorphogenesis, chloroplast biogenesis and maintenance, apical dominance, and senescence. In addition, this growth regulator is thought to antagonize BR""s ability to promote hypocotyl elongation and cotyledon de-etiolation.
In general, the invention features a substantially pure plant C-14 sterol reductase polypeptide. Preferably, the C-14 sterol reductase polypeptide includes an amino acid sequence substantially identical to the sequence shown in FIG. 14 (SEQ ID NO: 1); and is from a dicot (for example, a crucifer or a solanaceous plant), monocot, gymnosperm, or an alga.
In related aspects, the invention features purified DNA that includes a sequence encoding a C-14 sterol reductase polypeptide (for example, a sequence substantially identical to the DNA sequence shown in FIG. 14; SEQ ID NO: 2; or a DNA sequence that encodes a C-14 sterol reductase polypeptide which has an amino acid sequence substantially identical to that shown in FIG. 14; SEQ ID NO: 1). The invention also features a vector and a cell, each of which includes purified DNA encoding a C-14 sterol reductase polypeptide; and a method of producing a recombinant C-14 sterol reductase polypeptide involving providing a cell (for example, a plant cell) transformed with purified DNA encoding a C-14 sterol reductase polypeptide positioned for expression in the cell, culturing the transformed cell under conditions for expressing the DNA, and isolating the recombinant C-14 sterol reductase polypeptide. The invention further features recombinant C-14 sterol reductase produced by such expression of a purified DNA, and an isolated antibody that specifically recognizes and binds a plant C-14 sterol reductase polypeptide.
In addition, the invention features nucleotide sequences that hybridize to a C-14 sterol reductase gene (including the coding sequence of such a gene and its complement) and that encode a C-14 sterol reductase polypeptide. Furthermore, the invention includes oligonucleotide probes that detect a C-14 sterol reductase gene or functional equivalents thereof in a plant (for example, dicots (such as solanaceous and cruciferous plants), monocots, gymnosperms, and algae). Such probes are useful to isolate DNA sequences that encode C-14 sterol reductases from other plants. In one particular example, oligonucleotides may be designed based on a C-14 sterol reductase sequence disclosed herein and used as hybridization probes or as primers in polymerase chain reactions (PCR). Conserved regions in the C-14 sterol reductase gene are useful in the design of such primers to facilitate the recovery of C-14 sterol reductases from other related and unrelated plants.
In yet other related aspects, the invention features a transgenic plant (or seeds or cells thereof) containing DNA encoding a C-14 sterol reductase polypeptide integrated into the genome of the plant, where the DNA is expressed in the transgenic plant, resulting in the production of a C-14 sterol reductase polypeptide.
In still another aspect, the invention features a method for reducing the level of a plant C-14 sterol reductase polypeptide in a transgenic plant cell. This method generally involves expressing in the transgenic plant cell an antisense C-14 sterol reductase polypeptide nucleic acid sequence. In general, such an antisense C-14 sterol reductase nucleic acid sequence is encoded by a transgene integrated into the genome of the transgenic plant cell and is based on the nucleotide sequence that is shown in FIG. 14 (SEQ ID NO: 2) or FIG. 15. (SEQ ID NO: 3). In preferred embodiments, the plant cell expressing an antisense C-14 sterol reductase nucleic acid sequence is a dicot (for example, crucifer), monocot, gymnosperm, or algal cell. In yet other preferred embodiments, the method involves growing a transgenic plant from the transgenic plant cell, whereby the level of the C-14 sterol reductase polypeptide is reduced in the transgenic plant.
In other related aspects, the invention features a plant cell expressing an antisense C-14 sterol reductase nucleic acid sequence and a plant expression vector that includes an antisense C-14 sterol reductase nucleic acid sequence, where the antisense sequence is operably linked to an expression control region.
In another aspect, the invention features a method for increasing the level of a C-14 sterol reductase in a transgenic plant cell. This method involves expressing in the transgenic plant cell a C-14 sterol reductase polypeptide nucleic acid sequence. Preferably, the method utilizes a C-14 sterol reductase nucleic acid sequence that is substantially identical to the nucleotide sequence that is shown FIG. 14 (SEQ ID NO: 2). In preferred embodiments, the plant cell expressing a C-14 sterol reductase polypeptide nucleic acid sequence is a dicot (for example, a crucifer), monocot, gymnosperm, or algal cell.
In another aspect, the invention features a transgenic plant having a knockout mutation in DNA encoding a plant C-14 sterol reductase polypeptide. Such knockout genes are constructed according to conventional methods (e.g., Lee et al. Plant Cell 2: 415, 1990; Miao and Lam, Plant J. 7: 359, 1995).
By xe2x80x9cplant C-14 sterol reductasexe2x80x9d is meant an amino acid sequence that catalyzes the reduction of any sterol precursor having a C14=C15 double bond, for example, as described by Benveniste, Annu. Rev. Biochem. 37: 275, 1986. Preferably, such a polypeptide has an amino acid sequence which is at least 30%, preferably 40%, and most preferably 50% or even 80-95% identical to the amino acid sequence of the C-14 sterol reductase polypeptide shown in FIG. 14 (SEQ ID NO: 1). The length of comparison of amino acid sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably at least 35 amino acids.
By xe2x80x9cpolypeptidexe2x80x9d or xe2x80x9cproteinxe2x80x9d is meant any chain of amino acids, regardless of length or post-translational modification (for example, glycosylation or phosphorylation).
By a xe2x80x9csubstantially identicalxe2x80x9d polypeptide sequence is meant an amino acid sequence that differs only by conservative amino acid substitutions, for example, substitution of one amino acid for another of the same class (e.g., valine for glycine, arginine for lysine, etc.) or by one or more non-conservative substitutions, deletions, or insertions, located at positions of the amino acid sequence that do not destroy the function of the polypeptide (assayed, for example, as described herein).
Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group (University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705), BLAST, or PILEUP/PRETTYBOX programs). Such software matches sequences by assigning degrees of homology to various substitutions, deletions, and other modifications.
By xe2x80x9csubstantially pure polypeptidexe2x80x9d is meant a polypeptide preparation that is at least 60% by weight (dry weight) the compound of interest, for example, the C-14 sterol reductase polypeptide or C-14 sterol reductase-specific antibody. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
By xe2x80x9cpurified DNAxe2x80x9d is meant DNA that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5xe2x80x2 end and one on the 3xe2x80x2 end) in the naturally-occurring genome of the organism from which it is derived. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or that exists as a separate molecule (for example, a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences. It also includes a recombinant DNA that is part of a hybrid gene encoding one or more additional amino acids.
By a xe2x80x9csubstantially identicalxe2x80x9d nucleic acid is meant a nucleic acid sequence that encodes a polypeptide differing only by conservative amino acid substitutions, for example, substitution of one amino acid for another of the same class (e.g., valine for glycine, arginine for lysine, etc.) or by one or more non-conservative substitutions, deletions, or insertions, located at positions of the amino acid sequence that do not destroy the function of the polypeptide (assayed, for example, as described herein). Again, the encoded sequence is at least 30%, more preferably 40%, and most preferably 50%, or even 80 to 95% identical at the amino acid level to the sequence of FIG. 14 (SEQ ID NO: 1). Thus, when nucleic acid sequences are compared, a xe2x80x9csubstantially identicalxe2x80x9d nucleic acid sequence is one which is at least 30%, more preferably 40%, and most preferably 50%, or even 80 to 95% identical to the sequence of FIG. 14 (SEQ ID NO: 2). The length of nucleic acid sequence comparison will generally be at least 30 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides. Again, identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705).
By xe2x80x9cisolated antibodyxe2x80x9d is meant antibody that is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, antibody.
By xe2x80x9cspecifically bindsxe2x80x9d is meant an antibody that recognizes and binds a C-14 sterol reductase polypeptide but which does not substantially recognize and bind other molecules in a sample (e.g., a biological sample) which naturally includes a C-14 sterol reductase. An antibody which xe2x80x9cspecifically bindsxe2x80x9d a C-14 sterol reductase is sufficient to detect a C-14 sterol reductase product in such a biological sample using one or more of the standard immunological techniques available to those in the art (for example, Western blotting or immunoprecipitation).
By xe2x80x9can antisense C-14 sterol reductase sequencexe2x80x9d is meant a nucleotide sequence that is complementary to a plant C-14 sterol reductase messenger RNA. In general, such an antisense sequence will usually be at least 15 nucleotides, preferably about 15-200 nucleotides, and more preferably 200-2,000 nucleotides in length. The antisense sequence may be complementary to all or a portion of the plant C-14 sterol reductase mRNA nucleotide sequence, and, as appreciated by those skilled in the art, the particular site or sites to which the antisense sequence binds as well as the length of the antisense sequence will vary, depending upon the degree of inhibition desired and the uniqueness of the antisense sequence. By binding to the appropriate target sequence, an RNA-RNA, DNA-DNA, or RNA-DNA duplex is formed. A transcriptional construct expressing a plant C-14 sterol reductase antisense nucleotide sequence includes, in the direction of transcription, a promoter, the sequence coding for the antisense RNA on the sense strand, and a transcriptional termination region. Antisense C-14 sterol reductase sequences may be constructed and expressed as described herein or as described, for example, in van der Krol et al., Gene 72: 45, 1988; Rodermel et al., Cell 55: 673, 1988; Mol et al., FEBS Lett. 268: 427, 1990; Weigel and Nilsson, Nature 377: 495, 1995; Cheung et al., Cell 82, 383, 1995; and U.S. Pat. No. 5,107,065. In addition, C-14 sterol reductase antisense sequences are useful for the formation of triple helices, where the antisense sequence is bound to a DNA duplex. By binding to the target nucleic acid, C-14 sterol reductase antisense sequences can inhibit the function of the target nucleic acid. This results, for example, in the blocking of transcription, processing of poly A+ addition, replication, translation, or promoting inhibitory mechanisms of the cell, such as RNA degradation. The triple helix-forming and antisense C-14 sterol reductase sequences are useful for selectively suppressing certain cellular functions that are associated with C-14 sterol reductase activity.
By a xe2x80x9ctransformed cellxe2x80x9d is meant a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a DNA molecule encoding (as used herein) a C-14 sterol reductase polypeptide (for example, a substantially identical DNA encoding the C-14 sterol reductase shown in FIG. 14 (SEQ ID NO: 2)).
By xe2x80x9cpositioned for expressionxe2x80x9d is meant that the DNA molecule is positioned adjacent to a DNA sequence which directs transcription and translation of the sequence (for example, facilitates the production of, for example, a plant C-14 sterol reductase polypeptide such as the amino acid sequence shown in FIG. 14 (SEQ ID NO: 1)), or an RNA molecule (for example, an antisense RNA).
By xe2x80x9cpromoterxe2x80x9d is meant a minimal sequence sufficient to direct transcription. Included in the invention are promoter elements that are sufficient to render promoter-dependent gene expression controllable for cell-, tissue-, or organ-specific gene expression, or elements that are inducible by external signals or agents (for example, light-, pathogen-, wound-, stress-, or hormone-inducible elements); such elements may be located in the 5xe2x80x2 or 3xe2x80x2 regions of the native gene or engineered into a transgene construct.
By xe2x80x9coperably linkedxe2x80x9d is meant that a gene and a regulatory sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (for example, transcriptional activator proteins) are bound to the regulatory sequence(s).
By xe2x80x9ccruciferxe2x80x9d is meant any plant that is classified within the Cruciferae family as commonly described in, e.g., Gray""s Manual of Botany American Book Company, N.Y., 1950; Hortus Third: A Concise Dictionary of Plants Cultivated in the U.S. and Canada, Macmillan, 1976; or Simmons, N. W., Evolution of Crop Plants, 1986. The Cruciferae include many agricultural crops, including, but not limited to, broccoli, cabbage, brussel sprouts, rapeseed, kale, Chinese kale, cauliflower, horseradish, and Arabidopsis.
By xe2x80x9cplant cellxe2x80x9d is meant any self-propagating cell bounded by a semi-permeable membrane and containing a plastid. Such a cell also requires a cell wall if further propagation is desired. Plant cell, as used herein includes, without limitation, algae, cyanobacteria, seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores.
By xe2x80x9ctransgenexe2x80x9d is meant any piece of DNA that is inserted by artifice into a cell, and becomes part of the genome of the organism which develops from that cell. Such a transgene may include a gene which is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism.
By xe2x80x9ctransgenicxe2x80x9d is meant any cell that includes a DNA sequence which is inserted by artifice into a cell and becomes part of the genome of the organism which develops from that cell. As used herein, the transgenic organisms are generally transgenic plants and the DNA (transgene) is inserted by artifice into the nuclear or plastidic genomes.
Other features and advantages of the invention will be apparent from the following detailed description thereof, and from the claims.